Antitumor effects of flavopiridol on human uterine leiomyoma in vitro and in a xenograft model

Abstract

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21(cip/wafl) and p27(kip1) in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma.

Keywords: flavopiridol; leiomyoma; p27kip1; uterus; xenograft.

Figure 1.

Antiproliferative effect of flavopiridol in uterine leiomyoma cells. Growth inhibition in uterine leiomyoma cells treated with the indicated dose for 24 hours. Cell viability was measured by MTT assay and the results are expressed as a percentage of viable cells. Values are ±SD of 3 measurements, *P < .05. MTT indicates 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation.

Figure 2.

Inhibition of DNA synthesis in uterine leiomyoma cells by flavopiridol. Antiproliferative effects were determined after treating uterine leiomyoma cells for 24 hours with 100 and 200 nmol/L flavopiridol and BrDU labeling (10 µmol/L) for 24 hours. Results are expressed as a percentage of BrDU incorporation relative to control cells. Values are ±SD of 3 measurements, *P < .05. BrDU indicates 5-bromo-2′-deoxyuridine; SD, standard deviation.

Figure 3.

Effect of flavopiridol treatment on the cell cycle profile. After treatment with the indicated dose of flavopiridol for 24 hours, uterine leiomyoma cells were collected, fixed, stained with PI, and analyzed by flow cytometry. Values shown represent the number of cells in each phase as a percentage of the total cells. PI indicates propidium iodide.

Figure 4.

Effect of flavopiridol on cell cycle-related protein expression in uterine leiomyoma cells. Twenty-four hours after treatment, cell extracts were prepared and subjected to immunoblotting analysis. β-Actin was used as an internal loading control. Each band was quantified by densitometric analysis and presented in a bar graph.

Figure 5.

Histological evaluation of human uterine leiomyoma xenografts. Representative histological sections stained with hematoxylin and eosin (H&E). Immunohistochemistry using antibodies against α-smooth muscle actin (αSMA), desmin, estrogen receptor (ER) α, and progesterone receptor (PR) from matched tissue and cell graft. Original magnification was ×200.

Figure 6.

Effect of flavopiridol on xenografted tumors. A, Mice bearing established cell grafts were treated with flavopiridol at 5.0 or 7.5 mg/kg or DMSO control for 3 weeks starting on week 8. The graphs indicate the percentage of tumor growth in control and flavopiridol-treated groups. B, Representative image of control mice and mice treated with flavopiridol. C, Average tumor weights at the termination of the protocol in the control, 5.0, and 7.5 mg/kg of flavopiridol-treated groups. Values are ±SD of 3 measurements, *P < .05. DMSO indicates dimethyl sulfoxide; SD, standard deviation.